October 2009

October 19, 2009

There's a new edition of the JCB out today. We'll start our roundup with Tom Misteli's editorial on the recent JCB/New York Academy of Sciences meeting on the Cell Biology of Disease. Tom explains the rationale behind the meeting, much as he did in his introductory remarks to the conference. You can find our full coverage of the meeting by clicking here.

Elsewhere in this issue, our "In Focus" centers on the discovery by Canudas and Smith that cohesion between sister telomeres and centromeres is mediated by slightly different versions of the cohesin complex. Specifically, one of the cohesin subunits, Scc3, comes in two different flavors in vertebrate cells. Canudas and Smith show that Scc3SA1 binds telomeres together, while Scc3SA2 keeps sister centromeres close and, as you can read in my summary, telomere cohesion turns out to particularly important for the cell - defects in Scc3SA2 or its binding partner TIN2 result in DNA double strand breaks and telomere loss. Meanwhile, Adelfalk et al. show that a meiosis-specific cohesin called Smc1beta is essential for maintaining telomere integrity and attaching them to the nuclear envelope.

Our cover image this issue is taken from Koto et al.'s investigation of caspase regulation during development of the Drosophila sensory organ. Caspases aren't just involved in apoptosis, they can also play constructive roles in development - promoting the elongation of sensory organ shaft cells, for example. Naturally, caspase activity needs to be tightly regulated to allow their function for only as long as it takes to promote development without causing cell death. Koto et al. reveal that temporal differences in the expression levels of a caspase inhibitor called DIAP1 help caspases walk this fine line.

Tsuruta et al. show how a lipid kinase called PIKfyve helps neurons avoid cell death. Neuronal death can arise from overexcitation of their voltage-gated calcium channels (during a stroke for example). As explained in this summary, Tsuruta et al. show that sustained activation leads to the recruitment of PIKfyve to calcium channels at the membrane, where it generates the phospholipid PtdIns(3,5)P2. This, in turn, leads to the lysosomal targeting of the calcium channels, reducing calcium entry and cellular toxicity.

Next, we move from lysosome trafficking to melanosomal transport. Melanosomes are the specialized pigment granules of skin melanocytes that - similarly to lysosomes - arise from the endosomal system. Delevoye et al. show how two proteins, the clathrin adaptor AP-1 and the kinesin motor KIF13A, coordinate melanogenesis by positioning endosomes near to developing melanosomes and by sorting melanosome-specific cargo such as the enzymes that generate the melanin pigment. Moreover, Delevoye et al. provide some beautiful electron tomography to show that transport to the melanosomes is mediated by direct contact with tubules protruding from the endosomes - as in this image, where endosome tubules are in green and the melanosome is colored red. This paper will be the subject of our next biosights video, due in two weeks time but, in the meanwhile, you can read Lakkaraju et al.'s commentary on the article, which puts these findings into a nice perspective.

We also have a commentary (by Juliane Kellner and Pierre Coulombe) on a report by Vijayaraj et al. that keratins - the most neglected components of the cytoskeleton - have a surprising role in regulating protein synthesis. Mice lacking all their intermediate keratin filaments die in early embryogenesis due to severe growth retardation. It turns out that glucose transporters are mislocalized in the absence of keratins, leading to activation of the mTOR pathway and repression of protein synthesis. You can also learn more about this study in the latest edition of our biobytes podcast - also out today - in which Eun Choi speaks to the lead author Preethi Vijayaraj. And you can hear my interview with Greg Longmore on his lab's investigation of the antagonistic relationship between two rho GTPases in controlling cell shape that was published earlier this month. You can listen to biobytes by subscribing on iTunes, or by clicking here.

And don't forget to check out the rest of today's new articles by visiting our table of contents...

October 15, 2009

The JCB congratulates
Editor Elaine Fuchs, who will be the North American recipient of a 2010
L'Oréal-UNESCO Award
in the Life Sciences. Elaine is one of five scientists representing
five
continents who will be honored this year with the award, which
recognizes
exceptional women scientists. She will receive the award at a ceremony
in Paris
on March 4.

October 06, 2009

Time for a quick rundown of highlights from the latest issue of the JCB...

Our cover image this week is a section through a mouse whisker, stained in red for Merkel cells, which transmit mechanical stimuli to sensory neurons (stained in green). In other words, Merkel cells are involved in touch sensation, but the embryonic origin of these cells was unclear, since they have characteristics of both epidermal and neuronal cell types. In this issue, Van Keymeulen et al. demonstrate that they arise from epidermal progenitors during both embryogenesis and adult homeostasis. Moreover, deletion of a transcription factor called Atoh1 in these cells completely prevents Merkel cell development. You can read more in this summary, including why these findings may shed light on a particularly aggressive cancer that arises from Merkel cells.

Meanwhile, our In Focus for this issue centers on a study from Sivaramakrishnan and Spudich that reveals how multiple myosin motors coordinate their activities to transport vesicles along the actin cytoskeleton. The researchers use a combination of experimentation and computer modeling to show that even monomeric myosins (which would be completely unable to move cargo on their own) can join forces to carry the load.

Switching to mitosis, O'Connell et al. use a fascinating cell biological phenomenon called MUG to unpick the contributions that kinetochores and chromatin make to the assembly of the mitotic spindle. MUG stands for Mitosis with Unreplicated Genomes, and is induced by the prolonged treatment of cells with hydroxyurea. The fascinating thing is that these cells form a normal mitotic spindle attached to kinetochores and centromeric DNA aligned at the metaphase plate. Yet the rest of the cell's (unreplicated) chromatin is pushed out to the cell periphery. As you can read in this summary, O'Connell et al. use these cells to show that kinetochores alone can direct spindle assembly without the help of a Ran-GTP gradient generated from the bulk chromatin.

Meanwhile, McCleland et al. block DNA replication in early stage Drosophila embryos to demonstrate that S phase controls the speed at which the embryonic nuclei enter mitosis. Earlier this year, the researchers used the same experimental system to observe the contributions of cyclins to mitotic progression. I won't go into any more detail here, as Eun Choi has produced an extra special edition of biosights that covers both papers and features interviews with both Mark McCleland and Pat O'Farrell. Eun even made a trip to UCSF to film the researchers at work - you can see the video by clicking here, or by watching below:

Staying with flies, Shen and Ganetzky show how autophagy promotes Drosophila synapse development. You might think of autophagy as being a destructive pathway, but it actually plays a constructive role at neuromuscular junctions by counteracting proteosome-mediated degradation. Flies overexpressing the autophagy protein Atg1 have larger NMJs because they destroy an E3 ubiquitin ligase that would otherwise limit synaptic size. As I explain in this summary, the authors believe autophagy is a great way for neurons to regulate synaptic plasticity in response to many different environmental stimuli.

Finally for today, we have two papers that investigate the structures of protein particles transported back and forth along the length of cilia and flagella. Bidirectional transport of these particle trains is essential for assembling and maintaining flagella. Pigino et al. provide a beautiful electron-tomographic analysis of the "trains" of particles heading in both the anterograde and retrograde directions. Particle trains heading to the tip of the flagellum (indicated with a black arrowheads in the image to the right) are longer and narrower than trains returning to the organelle's base (white arrowhead).

How do cells control the size of cilia and flagella? Engel et al. demonstrate that trains traveling to the flagella tip are smaller in longer cilia. As the group's modeling shows, this may help the organelle to keep a consistent length, as the assembly rate will decrease as the cilia/flagella grows larger.

Plenty more in elsewhere in this issue. As always, you can find the full table of contents by clicking here.

October 02, 2009

In addition to each of its sessions onChromosomes, Cancer, and Stem Cells, the JCB Cell Biology of Disease Meeting had five powerful keynote addresses (two at the beginning and three at the end) that further highlighted exciting work underlying the cell biological basis of disease.

Opening Night

Our opening speaker on Wednesday evening was Ken Yamada(National Institutes of Health).Yamada discussed work by his group recently published in the JCB which uses micropatterning to examine the effects of extracellular matrix dimensionality on mechanisms of cell motility (Doyle et al., 2009). Yamada and his team noticed that cells traveling in 3D often follow single fibrils of matrix for extended periods of time, and wondered what cell migration would look like if cells were given single tracks of matrix to follow- a matix they refer to as one-dimensional. In a nutshell, cell migration in 1D recapitulates migration in 3D, while migration in 2D remains distinct from both.If a picture is worth a thousand words- our biosights videocaston this study speaks volumes, and will bring you up to speed in less than 8 minutes.

Live cell imaging has revealed key differences in cell morphology, adhesion, receptor usage, and signal transduction pathways on flat or voluminous matrices.For example, inhibition of myosin II doesn’t disrupt cells moving in 2D, but it does block migration in 3D and 1D.One debate central to 3D migration is whether proteases are required for cell migration or tissue invasion in vivo.Yamada’s group is now using fluorescence ratio imaging with a probe they’ve developed to detect metalloprotease activity in real time to look at adhesion dynamics and matrix degradation as cells migrate through 3D matrices.

Tissue organization during development is another fascinating way to study cell migration in a 3-dimensional context.

Yamada described experiments his group is using to study branching morphogenesis in vivo.It was fascinating to watch cells from a disrupted gland spontaneously aggregate and regenerate a new gland.A second surprise was revealed by live cell imaging.Although epithelial cells are commonly viewed as static in developed tissues, they are actually constantly moving, even in “fully developed” glands.Yamada is trying to figure out how clefts and buds, characteristic structures of branching tissues, form:What are the cell shape changes and the function of the matrix in forming these structures?He also described some preliminary work characterizing a protein that promotes cleft formation.

The next talk on opening night was given by Alan Hall, another member of the JCB Editorial Board.How cells establish polarity and directionality during migration is one focus of Hall’s work.Hall has a career’s worth of evidence to support a role for the Rho family GTPase Cdc42 in the regulation of cell polarity in tissue morphogenesis and in cell migration.

Cdc42 regulates the actin and microtubule cytoskeletons during polarized cell migration.Re-organization of actin is dependent on the Pak, an effector downstream of active Cdc42, while the Par6/aPKc pathway regulates microtubule polymerization.The cdc42/Par6/aPKC complex is asymmetrically distributed in polarized cells (e.g. during migration or cell division), and localizes to the apical domain of polarized epithelial cells.

Hall’s group used a 3D morphogenesis assay, similar to one described by Ken Yamada in the previous talk, to examine the function of Cdc42 in the regulation of epithelial cell polarity (See the biosights episode here).When embedded in extracellular matrix, CaCo2 epithelial cells form fluid-filled cysts, and have an established polarity by a two-cell stage. As cells continue to divide, the apical markers remain centralized, suggesting that the orientation of the mitotic spindle and/or the site of cytokinesis specifies polarity.To their surprise, depleting Cdc42 with siRNA didn’t prevent lumen formation altogether, rather Cdc42-depeleted cells form multiple lumens due to misorientation of the mitotic spindle (Jaffe et al., 2009).

But Cdc42 is just one of 18 Rho family GTPases expressed in mammalian cells – Hall reasoned that other GTPases may also contribute to cell polarity establishment or maintenance.In addition, Rho GTPases are regulated by more than 80 GTPase exchange factors (GEFs) and nearly 70 GTPase activating proteins (GAPs), which expands the candidate pool significantly.Therefore, when Hall’s group conducted a shRNA screen for GTPase-related proteins involved in establishing cell polarity and tight junction formation, they expected more hits than they got – which was a disappointing “zero”.However, a screen of more than 100 downstream Rho GTPase effector proteins turned up three candidates, Par6, Pak4, and aPKC – the same components important for establishing microtubule orientation and polarity during C. elegans embryogenesis.In the CaCo2 system, Par6 or aPKC depletion phenocopies Cdc42 depletion, resulting in multiple-lumen formation.However, depletion of aPKC also disrupts normal junction formation.Finding out how all of these components work together to coordinate polarity and tissue morphogenesis is an ongoing project in Hall’s lab.

The Final Session

The final three talks of the meeting brought Chromosomes, Cancer, and Stem Cells together in one impressive session that beautifully illustrated the themes of the conference.

Don Cleveland began the final session of the symposium by introducing what he calls “the centromere paradox”.Centromeres, as we learned from Iain Cheesemanand others, are the DNA sequences that link chromosomes to spindle microtubules. Although the function of the centromere is conserved throughout evolution, the actual DNA sequence of centromeres is not.

Cleveland and others have argued that the conserved element is an epigenetic demarcation.The nucleosomes that form at centromere DNA include a histone H3 variant, called CENP-A.These centromeric nucleosomes are therefore structurally divergent and are conformationally more rigid than conventional nucleosomes that contain Histone H3. The exact architecture of CENP-A nucleosomes, however, is still up for debate (see Furuyama and Hennikof, 2009 and a recent JCB feature for details).

To determine how the epigenetic mark for centromeres is replicated, Cleveland ’s group used SNAP-tagging to introduce a fluorescent substrate to detect when and where new CENP-A was synthesized.They found that new CENP-A was synthesized and incorporated into centromeric chromatin during G1 (Jansen et al. 2007). Cleveland therefore argues that propagation of centromeric chromatin requires exit from mitosis.

Now we know when CENP-A is synthesized, but how is it incorporated into nucleosomes?Cleveland’s group used TAP affinity purification to identify components of the CENP-A prenucleosomal complex, which is significantly different from histone-H3 containing nucleosomes, and found a chaperone HJURP that loads CENP-A onto chromatin during G1 (Foltz et al., 2009).

From CENP-A, Cleveland skipped through the CENP alphabet to CENP-E.CENP-E is an unusual kinesin required for correct chromosome alignment.Bound to kinetochores by its tail domain, its motor domain extends outwards from the chromosomes towards microtubules eminating from the spindle. Cleveland describes CENP-E as a “tenacious, floppy tortoise”- because it is a very slow motor (50 times slower than other motors), but it is very processive – it remains bound to microtubules for minutes at a time.

Removal or reduced expression of CENP-E results in chromosme non-disjunction and high rates of aneuploidy.In male mice heterozygous for CENP-E, the Y chromosome mis-segregates with high frequency.To determine whether aneuploidy caused by loss of CENP-E can beget tumorigenesis, Cleveland’s team injected CENP-E +/- MEFs into nude mice, and indeed sees an increase in tumor development. Cleveland is excited about the promise of CENP-E inhibitors as anti-cancer therapeutic agents.Unlike taxol, which inhibits mictrotubule depolymerization and therefore affects function of all cells, inhibitors of CENP-E would be extraordinarily specific for dividing cells.

After an energetic talk by Cleveland, no momentum was lost when Haifan Lin(Yale University) stepped up to the stage.The discovery of piRNAs has turned junk DNA into a goldmine for Lin.

Lin found that most piRNAs are coded by stretches of the “junk” DNA that makes up ~98% of the human genome.piRNAs are just one class of short RNAs, which differ from miRNAs and siRNAs because they bring their targets to Piwi proteins, not argonaute family endonucleases.PiRNAs also are distinct due to their size (They’re a little bigger than miRNAs), and their shear number.Lin’s lab has now identified more than 60,000 piRNAs in mouse testis, and 14,000 piRNAs in the fly genom

Identifiying the function of piRNAs is a daunting task since there are so many of them (Lin 2007).However, where they are located in the genome is revealing – many piRNAs appear to regulate epigenomic templating.

Piwi family members, known as Miwi in mice, are piRNA-specific proteins. In Drosophila, knockout of Piwi results in defects in ovarian stem cells, which tend to differentiate into two egg chambers, instead of one cell maintaining stem-cell capabilities for self-renewal.In a yeast two-hybrid screen to identify other Piwi interacting proteins, Lin identified the heterochromatin protein 1A.HP1A controls heterochromatin silencing and molds chromatin structure through interactions with Histone H3K9.HP1A binding to chromatin requires Piwi complexed to piRNA, suggesting that piRNAs may target Piwi and chromatin remodeling proteins to specific areas of the genome.

Joan Massagué (Memorial Sloan Kettering Cancer Center) is something of a cellular archeologist.Many metastatic cancers have preferential sites they colonize, for breast cancer cells, those sites include bone, lung, and brain.In an ambitious comparative genomics project, Massagué isolated cells from each of these sites and compared them to cells isolated from the primary tumor, as well as healthy breast tissue.The process of metastasis itself, he argues, may define the progression of acquired traits that make invasion, survival and colonization of tumor cells in alternate tissues possible.

When breast cancer cells reach bone, they influence the behavior of native cells (Lu et al., 2009).Breast cancer cells induce osteoclasts to proliferate, differentiate, and resorb bone matrix. All of this activity releases additional growth factors that the cancer cells use to further proliferate (e.g. TGF-b, IGF-1).Breast cancer cells derived from bone metastatic sites express a distinct set of genes, including MMP1, a metalloprotease associated with invasiveness, ostopointin, a protein that regulates osteoclast function, and IL-11, a cytokine that induces osteoclastogenesis.

The gene set expressed by breast cancer cells isolated from lungs have yet another distinct set of genes.The lung metastasis signature identified by Massagué includes nearly 20 genes expressed in estrogen receptor negative tumors associated with high potential for relapse of lung metastasis. These genes include Cox2, a regulator of ROS production and inflammation, and Angiopoietin-like 4, important for breast cancer cell extravasation (Minn et al., 2005; Gupta et al., 2007; Padua et al. 2008).

When compared to cells derived from the primary tumor, Massagué was surprised to find that Angptl4 was also expressed there.Although Angptl4 doesn’t necessarily confer a growth advantage to cells in the primary tumor, when these cells reach the lung, it helps them to invade lung tissue by reducing cell-cell adhesions between pulmonary endothelial and epithelial cells –increasing their ability to invade into/through the epithelial layer.

The prominence of breast-cancer derived metastasis to the brain is more than ten times the incidence of primary brain tumors.Brain metastasis results in neurological disability, the tumors are resistant to treatments, are predominantly lethal, and poorly understood.Massagué found that Breast cancer cells positive for BrMS are more likely to invade brain tissue.A subset of genes are shared between lung and brain-metastatic cells, including Cox2 and Angplt4, which are both important for crossing the blood-brain-barrier.In addition- brain-tropic cells express ST6GalNac5, a sialyl transferase normally only expressed by brain-derived cells.

October 01, 2009

Prevention, diagnosis, and treatment of cancer remains a challenging scientific and medical problem.Our former Editor-in-Chief, Ira Mellman, opened the third session of the conference, focused on cancer on Friday morning with a discouraging figure. While the mortality rates from heart disease, stroke, influenza, and pneumonia have declined dramatically over the past 50 years, mortality due to cancer has changed little.Mellman stressed that ‘translational’ cell biology is a two-way street, and there is much to be gained from the application of cell biological-based theories to therapeutic practices as well as understanding the mechanisms of clinical observations at the cell-biological level.

Mutations in the HER/EGF receptor gene family have been linked to human cancer. Monoclonal antibody therapeutics have been used clinically (e.g. Erbitux(Imclone), Vectibix(Amgen), and Herceptin(Genentech)), but the mechanism of action for all of these reagents – or why they sometimes fail- is not entirely clear.

Signal transduction initiated by Her1/EGFR has been studied extensively, and the literature repeatedly iterates the dogma: EGF binding induces receptor dimerization and activation.A high resolution structure of Her1/EGFR was recently published (Jura et al., 2009).Indeed, ligand binding to the EGFR is predicted to change its confirmation from a closed to an open configuration, extending the intracellular dimerization loop to allow close apposition of receptor pairs for cross-phosphorylation and receptor activation.

However, molecular imaging studies are providing new insights into how EGF sets its receptor going.Using TIRFM, Mellman’s group at Genentech is following movement of the EGFR on the cell surface at the single molecule level.Labeling the receptor with quantum dots allows the receptors to be tracked for extended periods of time in live cells. The analysis revealed that receptors continuously
and reversibly form dimers from monomers in the absence of ligand, with
dimer formation being favored in the cell periphery -- despite an
apparently even distribution of total receptors across the cell surface.EGF binding requires dimerization competent but not activation competent EGFR, suggesting that dimerization of the receptor somehow favors ligand binding.Mellman proposes that spontaneous dimerization primes the receptor for ligand-induced signaling.The receptor predominantly exists as closed, low affinity monomers which “flicker” into inactive, higher affinity dimers primed for ligand binding and receptor activation.Mellman hopes that similar studies in the future will be useful for understanding how anti-EGFR therapeutics affect function and regulation of this cancer-promoting receptor.

Trotman continued the trend of overturning the textbook version of cancer mechanisms.The textbooks all say that it takes two hits – or multiple mutations - to make a tumor.Trotman’s transgenic mice say it takes just one thing: active Akt.

Akt, a kinase that regulates all insidious cancer behaviors – proliferation, survival, polarization, and migration, is localized to the plasma membrane through association with the phospholipid PIP3.In 2003, Trotman and colleagues generated mice which express varying levels of PTEN, a phosphatase that converts PIP3 to PIP2, and thus inactivates Akt.They found that even a 30% reduction in PTEN levels resulted in a correlative increase in phosphorylated Akt – and formation of prostate tumors (Trotman et al., 2003).

In 2006, Trotman found that tumor incidence also increased in mice lacking the tumor suppressor PML (Trotman et al. 2006).While Akt accumulates in the cytoplasm in PTEN-deficient cells, in PML-null cells it ends up in the nucleus where it inhibits pro-apoptotic and cell-cycle arrest programs.PML, he showed, is required to recruit the PP2A phosphatase to nuclear bodies to turn off Akt and suppress uninhibited cell growth.

Using a search for proteins that favor the same PIP3-enriched neighborhoods as Akt, Trotman has now discovered a third phosphatase that regulates Akt. Relying on a tried-and-true approach, Trotman’s group has knocked the gene out in transgenic mice to see if they, too, will develop prostate tumors due to over-active Akt.

As an additional resource, Trotman has also joined forces with the MSKCC oncogenome project, which has obtained more than 150 primary tumor samples, including 34 metastatic biopsies.The samples have been analyzed for alterations in Akt phosphatases by DNA copy number, mRNA expression levels, and immunohistochemistry. In these samples, there is frequent decrease in expression of PTEN.However, in the metastatic samples, both PTEN and the newly identified Akt-phosphatase seem to be missing. Understanding how loss of multiple phosphatases contributes to invasive phenotypes is an ongoing focus of research for Trotman’s group. For more information on Lloyd Trotman, see the P&I published in JCB last year.

Maria Blasco (CNIO, Madrid): The Long and the Short of iPS reprogrammingThe next speaker was JCB Editorial Board Member Maria Blasco.Blasco’s recent studies address the regulation of telomere length and its relationship to stem cell proliferative capacity and aging.With every cell division, the telomeric repeats at the tips of chromosomes are shortened.It’s the job of the shelterin complex, which binds to telomeres, to prevent chromosome instability and to compensate for loss of telomere length.Mutations in shelterin complex components are associated with rare, human stem cell diseases which typically manifest as bone marrow failure, aplastic anemia, pulmonary fibrosis, and skin hyperpigmentation.

Normally, stem cells have long telomeres, however shortened telomeres are coincident with cells that leave the stem cell niche and differentiate.In aged animals, the length of telomeres is generally decreased- even in stem cells.Increasing telomerase activity in transgenic mice increased the animal’s life span, presumably through delay of telomere shortening.

Blasco wondered what impact telomere shortening would have on stem cell function.In mice missing the telomere component TERF1, mobilization of epithelial stem cells in response to TPA treatment is decreased, making the skin more resistant to carcinogenic treatment. However, these mice trade resistance to cancer for premature aging (Martinez et al. 2009). The mice also have a hyperpigmentation phenotype, and so mimic many of the characteristics typical of the human pathologies associated with loss of TERF function.

What about reprogrammed, or induced pluripotent stem (iPS) cells?Blasco’s team recently showed that expression of the iPS inducing transcription factorsKlf4, Oct3/4, Sox2, and myc,“rejuvenated” telomeres.Blasco also noted that the shorter telomeres are, the harder it was to get cells to re-program.However, the decreased re-programming efficiency could be rescued by introduction of telomerase (Marion et al. 2009).The DNA damage response factor p53 also was a limiting factor for reprogramming efficiency, which initiates programmed cell death pathways in response to telomeres that have become too short.Blasco’s team showed that loss of p53 suppressed the accelerated aging phenotypes observed when there are defects in shelterin complex function and telomere protection or extension.

Muthuswamy uses a 3D culture system to approximate acinar formation of human breast tissue in vitro.He noticed that in addition to inducing rapid cell proliferation, expression of the breast-cancer inducing gene ErbB2 also disrupted the normal morphogenesis of these structures, which formed multiple, lumen-free acini mimicking the pre-malignant state observed in cancer patients.However, when proliferation was induced by over-expression of cyclin D or inactivation of Rb, hyperplastic growth occurred – but the structure of the tissue was maintained. He was quick to point out that in 2D, under the same circumstances, no phenotype is observed.

Muthuswamy’s curiosity about how disruption of normal tissue morphogenesis affects tumor development has been a source of fixation throughout his career (Be on the look out for a P&I feature on Muthuswamy in the October 19 Issue of the JCB, Feigin and Muthuswamy 2009). His suspicion that defects in regulation of cell polarity or a normal morphogenetic program may be an important component of metastatic tumor growth was confirmed by a recent set of experiments, which investigated the function of Scribble, a scaffolding protein that localizes to cell-cell junctions and is downregulated in certain cancer types.

Decreasing expression of scribble disrupts polarity and organization of epithelial cells grown in 3D culture.Loss of scribble also blocks apoptosis during lumen formation, but does not have an effect on proliferation.In vivo, loss of scribble disrupts mammary gland morphogenesis.To find out what happens if you combine scribble deficiency with a proliferation-inducing oncogene, Muthuswamy expressed c-Myc in scribble knockout cells.The result was strikingly similar to the neoplastic growths observed when ErbB2 is over-expressed (Zhan et al., 2008).

Alissa Weaver (Vanderbilt University): When it comes to invasion, tumors are inflexible.

Tissue composed of tumor cells is often more rigid than healthy tissue, perhaps due to increased cell packing, inflammatory or scar tissue infiltrates, or excess deposition of extracellular matrix.In humans, this kind of dense tissue is associated with an increased risk of invasive breast cancer, which Weaver studies in her lab.

Weaver wondered if the stiffness of the extracellular matrix could influence tumor invasion by modifying the formation or function of invadopodia- specialized cellular protrusions that promote localized matrix degradation.Formation of invadopodia correlates with invasion through basement membrane and tumor metastasis in nude mouse models.

Weaver’s team in fact found that increased matrix density can increase both the number and degradative capacity of invadopodia in vitro (Alexander et al., 2008).The ability to “sense” tension, through integrin-mediated activation of Myosin II and MLCK is important for invadopodia formation. Other candidates for mechanosensory transduction are p130 Cas and the focal adhesion kinase, FAK.Phosphorylation of both molecules is increased in invadopodia, but not in the presence of the myosin inhibitor blebbistatin, which inhibits tension signaling.

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